The polyol pathway enzyme aldose reductase (AR) has been implicated in several pleiotrophic complications of diabetes. In animal models, AR inhibitors (ARI) prevent or delay multiple diabetic complications. However, the clinical efficacy of ARI remains uncertain, and the physiological role of AR is unclear. Our results during the past funding period show that hydrophobic aldehydes, such as those derived from lipid peroxidation are reduced more efficiently by AR than is glucose; raising the possibility that AR normally participates in the detoxification of lipid-derived aldehydes, and in diminishing oxidative stress. Our central hypothesis is that by altering the cellular redox state, and inducing post-transnational modifications, prolonged diabetes perturbs the antioxidant role of AR, so that it contributes to, rather than combats, the oxidative effects of high glucose. To test this hypothesis, we will investigate the post-translational changes in AR under hyperglycemic vs. normoglycemic conditions in human lens epithelial cells (HLEC), vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC). Based on our results showing that AR could be nitrosated or glutathiolated in vitro, we will determine how an increase in nitric oxide (NO) synthesis, nitrosothiols, and NO donors affects cellular AR activity (Aim 1). By electrospray mass spectroscopy of immunoprecipitates, we will identify covalent changes in AR in cells or tissues exposed to hyperglycemia or diabetes (Aim 2). The role of AR in regulating endogenous oxidative stress will be examined within the context of tumor necrosis factor-alpha (TNF-alpha)-induced signaling, which is increased in diabetes and is mediated by reactive oxygen species. We will test whether inhibition of AR prevents the activation of NF-kappaB by TNF-alpha and so leads to a decrease in TNFalpha-induced VSMC growth and HLEC or VEC apoptosis (Aim 3). We will examine whether the basal activation of NF-KappaB by high glucose is prevented by ARI, and whether they prevent the mitogenic and apoptotic effects of TNF-alpha in high glucose (Aim 4). These studies should lead to the identification of a novel mechanism by which ARI prevents diabetic complications, and will provide a better understanding to the regulation and the role of AR in normoglycemic conditions. Our investigations will also help in evaluating the long-term risks or benefits of anti-AR therapy.